Separation of heteromeric potassium channel Kcv towards probing subunit composition-regulated ion permeation and gating

The chlorella virus-encoded Kcv can form a homo-tetrameric potassium channel in lipid membranes. This miniature peptide can be synthesized in vitro, and the tetramer purified from the SDS–polyacrylamide gel retains the K+ channel functionality. Combining this capability with the mass-tagging method, we propose a simple, straightforward approach that can generically manipulate individual subunits in the tetramer, thereby enabling the detection of contribution from individual subunits to the channel functions. Using this approach, we showed that the structural change in the selectivity filter from only one subunit is sufficient to cause permanent channel inactivation (“all-or-none” mechanism), whereas the mutation near the extracellular entrance additively modifies the ion permeation with the number of mutant subunits in the tetramer (“additive” mechanism).